V-OLA: point-of-care HIV viral load monitoring and drug resistance testing

摘要 我们将开发第一种检测病毒载量（VL）和HIV耐药性（DR）的检测方法， 低资源环境（LRS）。这项新的测试是基于多年来开发和测试 用于HIV DR的寡核苷酸连接试验（奥拉），以及我们最近开发的OLA-Simple - a实验室试剂盒 这减少了奥拉的时间和复杂性，但仍然需要实验室设备，实验室单向 防止扩增子污染的工作流程，以及多个步骤的实际操作。建议的V- 奥拉测试是一个重大的进步，使用创新的技术协调，以提供一个低成本， 适合艾滋病毒患者管理的精简测试套件。我们将开发快速样品制备技术， 在临床相关阈值下的全血半定量VL测量（当前WHO截止值为 治疗失败>1000拷贝/mL），以及用于识别HIV DR突变的集成检测盒（目标是 对于VL >1000拷贝/mL的患者检测10%突变体）。在目标1中，我们将开发化学和样品 用于HIV RNA分离的制备形式与快速VL检测兼容。在目标2中，我们将开发VL测试 使用半定量等温扩增，包括简单的低成本加热块和检测器。在目标3中， 我们将开发核心奥拉化学，包括DR靶区域的等温扩增， 单链扩增子、用于扩增干试剂和奥拉;我们将对不同的HIV亚型进行检测。在 目的4：建立一套完整的DR检测装置，包括等温扩增和连接 侧流条带检测。在目标5中，我们将联合收割机将VF和DR测试组合到一个协调系统中，包括 改进硬件，扩大一次性套件的制造规模，并开发一个数字辅助系统-称为 水族馆-提供分步指导方案、样本跟踪、结果解释和预定 质量控制测试。我们将在库存HIV血浆样本（HIV-1亚型A、C、D和AE）上检测V-OLA。 此级别的测试旨在为性能、可用性、 和执行

ABSTRACT We will develop the first test for detection of viral load (VL) and HIV drug resistance (DR) that is appropriate for low-resource settings (LRS). This new test is based on years of experience developing and testing the Oligonucleotide Ligation Assay (OLA) for HIV DR, as well as our recent work to develop OLA-Simple – a lab kit that reduces the time and complexity of OLA, but still requires laboratory equipment, laboratory one-way workflow to prevent contamination by amplicons, and hands-on operation of multiple steps. The proposed V- OLA test is a major advance over OLA-Simple that uses innovative technologies coordinated to give a low-cost, streamlined test suite appropriate for HIV patient management. We will develop rapid sample preparation from whole blood, semi-quantitative VL measurement at clinically-relevant thresholds (current WHO cut-off for treatment failure is >1000 copies/mL), and an integrated test cartridge to identify HIV DR mutations (goal to detect 10% mutant for patients with VL >1000 copies/mL). In Aim 1, we will develop chemistry and sample preparation format for HIV RNA isolation compatible with a rapid VL test. In Aim 2, we will develop the VL test using semi-quantitative isothermal amplification, including a simple low-cost heat block and detector. In Aim 3, we will develop core OLA chemistry including isothermal amplification of DR target regions, ligation on single- stranded amplicon, and dry reagents for amplification and OLA; we will test assays on different HIV subtypes. In Aim 4, we will develop the integrated DR detection device, including isothermal amplification and ligation with lateral flow strip detection. In Aim 5, we will combine the VF and DR tests into a coordinated system including refining hardware, scaling up fabrication of kit disposables, and develop a digitally-assisted system – called Aquarium – to provide step-by-step guided protocols, sample tracking, results interpretation, and scheduled quality control tests. We will test V-OLA on banked HIV plasma samples (HIV-1 subtypes of A, C, D, and AE). This level of testing is intended to provide preliminary evidence for in-depth studies on performance, usability, and implementation.